How Much Do You Know about Co-Immunoprecipitation?

Posted by Ellen Burns on January 7th, 2020

Proteins are executors of molecular functions that regulate all biological systems in the cell. However, usually they are not "alone", and the vast majority of functional proteins interact with other proteins to regulate life processes. Below we give an introduction to a technique commonly used in protein interaction studies, Co-immunoprecipitation (Co-IP).

Principle of the experiment: Co-IP uses the principle of specific immunobinding between antigen and antibody to add specific antibody to the cell lysate and precipitate the antigen and the protein bound to the antigen. Immune complexes can be used to verify the interaction between antigen and other proteins by western blot, or by mass spectrometry to detect the binding protein members of antigen.

Experimental procedures: How is the Co-IP experiment done? In a nutshell, the Co-IP experiment probably includes the following processes:

Construct the expression vector and transfect the cells (For this step, the bait protein is exogenous, and the endogenous bait protein directly enters the next step); Collect the cell lysate under mild conditions; Incubate the cell lysate with the antibody, and wash away the unbound protein; Elute the bait protein and the bound interacting protein complex with the appropriate eluent; Analysis by western blot or mass spectrometry.

Advantages and disadvantages of Co-IP technology:

Advantages: (1) The obtained interacting proteins naturally interact with bait proteins in cells, which is in accordance with the real physiological situation in vivo; (2) the experimental conditions are mild and can avoid artificial effects; (3) native state interacting protein complexes can be isolated.

Disadvantages: (1) Not applicable to the study of protein interactions with weak binding or transient binding.

The effect of the Co-IP experiment is affected by a variety of factors, and the following points should be noted:

1. The first is the quality of the antibody, which requires the use of IP grade antibodies;

2. Second, the number of cells, one Co-IP experiment recommends using two 10 cm dishes of cells grown to 80% confluence;

3. Cells need to use a mild lysis solution to extract proteins to prevent degradation and artificial modification;

4. Always set a control. Normally, the endogenous protein can be used as a control with Nomal IgG, and the exogenous protein can be transfected into cells with an empty vector as a control.